The understanding of the molecular mechanisms of the differentiation of neurons during development is of fundamental importance in neuroscience. We have used two neuronal hybridomas, NCB-20, and NG108-15 cells, to study the receptor regulation induced by two differentiation promoters, dibutyryl cAMP (Bt2 cAMP) and butyrate. Exposure of cells to Bt2cAMP caused a time and dose dependent decrease in the binding to muscarinic acetylcholine, adrenergic alpha2 and opioid delta receptors. The loss of muscarinic cholinergic receptor site assessed by using 3H-QNB as the binding ligand was about 40% of the control; this decrease was associated with an attenuation of the carbachol-induced phosphoinositide hydrolysis. The maximal decreases of adrenergic alpha 2 and opioid delta receptor binding were about 80% when 3H-clonidine and 3H-D-Ala-D-Leu- enkephalin (DADLE) were used as their respective ligands. In contrast, butyrate induced a time and dose dependent increase in the opioid receptor binding sites. The maximal increase was about 300% of the control when 1 mM of butyrate was added to the culture for 72 hrs. The up-regulation of opioid receptor resulted in an increased potency of DADLE in inhibiting adenylate cyclase activity. Similar butyrate treatment up-regulated muscarinic receptors by 100% and increased carbachol-induced phosphoinositide by about 200%. Long term butyrate treatment on NG 108-15 cells did not change muscarinic and opioid receptor binding but induced a 100% increase of the density of alpha 2- adrenergic receptors. Butyrate treatment induced cell morphological changes which were distinct from those induced by Bt2cAMP. These results suggest that these two differentiation agents can either up-regulate or down-regulate neurotransmitter receptor proteins. Moreover, up-regulation of neurotransmitter receptors induced by butyrate may involve concerted interactions of genetic factors derived from both parents of these two neurohybrids.